Radiopharmaceutical solutions with advantageous properties

ABSTRACT

Methods and solutions are describes whereby radiopharmaceutical radium-224 solutions are added complex for scavenging daughter nuclide, which may enhance the overall targeting and reduces uptake of daughter nuclide in normal tissues and cells. This is accomplished without reducing the bone targeting ability of radium. The methods and solutions are convenient to use since the complexation of daughter nuclide can be done in situ in a ready to use radiopharmaceutical solution or, depending on the activity concentration, in a simple kit format by mixing two solutions and store the mixture for typically 1 minute to a few hours before administration to a patient. The use of targeted chelate scavengers for  224 Ra daughter nuclide opens up the possibility for using  224 Ra based solutions for medical treatments.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a radiopharmaceutical solutioncomprising ²²⁴Ra and a complex capable of scavenging ²¹²Pb and/or ²¹²Bi.This solution can be used for medical purposes, including treatment ofcancer. Further aspects of the invention relates to kits and methods forproviding specific solutions.

2. Description of the Related Art

Targeted alpha particle radionuclide therapy holds promise astherapeutic modality against malignant and non-malignant diseases.Alpha-emitting radionuclides are highly cytotoxic and the alphaparticles produced are of high linear energy, which is delivering a highamount of ionization over a short range, causing destruction of DNA by ahigh degree of irreparable double strand breaks.

Thus, it is important when using alpha emitting radionuclides that theydo reach the target and are not released from the targeting compound andthat they do not produce longer lived daughter nuclides that diffuseaway from the mother nuclide, since this can cause toxicity in remotetissues.

There are relatively few alpha emitters considered for medicalapplications. It is a challenge in the field to find a radionuclide withappropriate half life, decay properties, chemical properties anddaughter products that are suitable for development of medicaltreatments.

Radium was very important for the development of radiochemistry sciencesand was also used by the pioneers of radio-oncology to treat cancer withbrachytherapy (, that is radiation emitting needles or seeds placedwithin or nearby tumors. Initially ²²⁶Ra with a half life (t_(1/2)) of1600 years was used. Later on ²²⁴Ra (t_(1/2)=3.66 days), as dissolvedradium chloride injectates, was used for several decades in Germany forthe palliative treatment of ankylosing spondylitis (AS) because of thenatural bone-seeking property of this, the heaviest of the alkalineearth elements.

Although reintroduced after the development of improved purificationmethods for a brief period about year 2000, its use was finallyabandoned partly due to fear of late effects and the appearance of newtreatment options for AS. Therefore ²²⁴Ra is not in use as aradiopharmaceutical today and is not considered among the plausiblecandidates for alpha therapy by leading experts in the field.

There is, however, research going on using ²²⁴Ra loaded wires for localintratumoral brachytherapy/radon diffusion therapy but they are notusing aqueous 224Ra solutions for therapy.

Recently, another radium isotope, ²²³Ra, in the form of a dissolved saltinjectate, has been granted market authorization as a treatment againstskeletal metastases from castration resistant prostate cancer.

When comparing ²²³Ra (t_(1/2)=11.4 days) with ²²⁴Ra (t_(1/2)=3.6 days)both have, in principal, relevant half-lives for radiopharmaceuticaluses allowing centralized production and shipment to the end user andboth have three alpha emitting progenies in their decay chains and theseries are producing a similar amount of alpha particles (FIGS. 1 and 2)with total alpha energy of about 26-28 MeV for the respective chains.

When comparing the decay chains, ²²³Ra progenies of the Rn, Pb and Bielements have significant shorter half lives reducing the problem ofdaughter nuclide uptake in non-target cells and tissues. Thesedifferences are particularly important for the lead progenies as thesecan accumulate in hematopoietic cells and tissues and in kidneys,respectively. In the ²²³Ra series ²¹¹Pb (t_(1/2)=36.1 min) would causemuch less normal tissue exposure compared with ²¹²Pb (t_(1/2)=10.6hours) from the ²²⁴Ra series. Unless ²²⁴Ra is purified from ²¹²Pb, shortbefore injection, ²²³Ra would have significantly less normal tissueexposure from progenies. Such purification is impractical since it wouldrequire laborious procedures to be performed at the hospital where theproduct is being used or that the production and use is beinggeographically restricted. That is why the time frame for use of ²²⁴Rathat was previously supplied by Altmann Terapie, Salzgitter, Germany,for AS, was of only 6 hours. It could be used 3 hours before or threehours after the calibration time point. Probably to a significant extentbecause of this short product shelf life (in addition to increasedcompetition with the new drugs for AS) and the thereby logistics andsupply constraint the product has been discontinued.

As of currently, there is no use of ²²⁴Ra solution for injection topatients. Instead, there is in development ion exchanger based ²²⁴Ragenerators for the extraction of ²¹²Pb for the use of ²¹²Pb inradioimmunotherapy. Lead-212 is itself a beta-emitter but decays to thealpha-emitter ²¹²Bi and is therefore considered suitable as an in vivogenerator for alpha particle therapy.

Thus, currently ²²⁴Ra is considered merely as a generator nuclide forthe medically useful ²¹²Pb. Because of the relatively short half life of²¹²Pb it is expected to be best suited in treatment againstcompartmental disease where the radioimmunoconjugate is injecteddirectly into the region, e.g., intraperitoneal (i.p.) cavity where ahigh concentration of product may target malignant ascites andmicrometastases within the cavity. The half life of 10.6 hours of ²¹²Pbmay be of benefit as only low amount leaks out from the i.p. cavitybefore the radioactivity has decayed.

When considering radium for therapy against bone diseases, it should bekept in a cationic state since this will ensure that that radium, as theso called “volume-seeker” it is, will be built into the bone mineralscausing retention of the daughter nuclides. This is particularlyimportant with ²²⁴Ra since some of the daughter nuclides, in particular²¹²Pb, have substantial half-lives allowing trans-organ redistributionif let free in physiological liquids like blood, saliva or lymphaticliquid. Table 1 (FIG. 4) lists the main radiation of the decay chain of²²⁴Ra.

A monograph about the use of ²²⁴Ra in ankylosing spondylitis weredeveloped by the German health authorities about 10 years ago whenAltmann Terapie (Salzgitter, Germany) made an effort to re-introduce²²⁴Ra solution as a treatment against ankylosing spondylitis based on apatented production method yielding a highly purified product.

In the decay chain of ²²⁴Ra the daughter product ²¹²Pb (t_(1/2)=10.6hours) is produced. It has a different biodistribution compared to the²²⁴Ra mother nuclide when co-injected in patients. This causes lessinitial activity in the target tissues and more activity in thenon-target tissues like blood cells, in particular hematopoietic cellsand tissues, and bone marrow and kidneys. The number of ²¹²Pb atomscompared with ²²⁴Ra in a product in radioactive equilibrium is less than14%. But because ²²⁴Ra rapidly transfer from blood to the skeleton or isexcreted and ²¹²Pb is substantially retained in hematopoietic cells andtissues, the toxicological impact of ²¹²Bi is important. The only way tosolve this problem by current knowledge in the field would be to use²²⁴Ra short time after purification as suggested, that is, before asignificant in-growth of ²¹²Pb has taken place.

The use of scavenger for daughter products in experimentalradiopharmaceutical research has been described: Jones et al (1996)studied oral administration over several days of the non-targeteddithiol chelating agents 2,3-dimercapto-1-propanesulfonic acid (DMPS)and meso-2,3-dimercaptosuccinic acid (DMSA) to improve the clearance of²⁰⁶Bi from kidneys in mice and found improved kidney clearance withDMPS. Their aim was to use oral chelate as potential adjuvants to reduceor prevent radiotoxicity in anti-interleukin-2 receptor (IL-2R) ²¹²Pb or²¹²Bi alpha-radioimmunotherapy. Jaggi et al., (2005) used oral chelationtherapy to reduce renal accumulation of ²¹³Bi produced from ²²⁵Ac. Theirgoal was to increase excretion of the undesired daughter product. Theydid not add the chelator to the radiopharmaceutical but merely describeits use as oral medication in the drinking water before and afterinjection of radiopharmaceutical.

In terms of bone therapy, ²²³Ra is considered more appropriate than²²⁴Ra because ²²³Ra have daughter nuclides with much shorter half-livesand thus, less problem of relocalization. Radium-223 has recently beenapproved for the therapy of patients with hormone refractory skeletalmetastases from prostate cancer.

Dissolved ²²⁴Ra salt has previously been tested in cancer therapy butwas abandoned because of unfavorable properties and ineffectiveness. Itwas stated that because of the short half life of ²²⁴Ra and its injecteddaughters, the soft tissue(s) are irradiated. In other words in the caseof ²²⁴Ra the half life of the daughters, in particular ²¹²Pb, arerelatively long compared to the mother nuclides and more soft tissueexposure occurs. Therefore it is known in the field that ²²⁴Ra hasunfavorable daughter nuclide restricting its use in radiopharmaceuticalsolutions. Also, in recent review by senior experts in the field, ²²⁴Rawas not listed among the candidate radionuclides considered for alphaparticle emitter radiopharmaceutical therapy.

The concept of circulating tumor cells (CTC) has lately receivedconsiderable attention as CTC may play a critical role in thedevelopment of tumor metastases. It is known that cancers that produceskeletal metastases like e.g., prostate-, breast-, lung- and multiplemyeloma cancers may have a few viable circulating cancer cells in theblood which may have been shed from the primary or metastatic tumors.This means that even if the bone metastases are treated new lesions canbe formed by the settlement of CTC's in the bone or other tissues.

Radium-223 used against bone metastases today is a pure bone-seeker anddo not address the problem of CTC's. Therefore there is a need in thefield of alpha pharmaceutical bone therapeutics of a product that canalso address CTC's.

The generation of daughter nuclides both in the injectates and in vivois a potential problem for ²²⁴Ra and to a lesser extent ²²³Ra as thefirst progeny in the two decay series for both is radon which is highlydiffusive. However literature data indicate that this is less of aproblem when generated in vivo since radium is a bone volume-seeker andis embedded in the bone matrix. It also helps out that the uptake inskeleton of intravenous radium occurs almost instantly and intestinal,and to a less degree urinary, elimination happens rapidly, leading to anelimination from the blood within minutes after injection. It should bementioned that urinary elimination is probably more pronounced inrodents compared to humans were fecal elimination is the main route. Asreported by Nilsson et al (2005), a reduction of 88% of radium in bloodat 10 minutes after injection occurs. When considering radium localizedin the skeleton, it was for ²²³Ra reported equilibrium of ²¹¹Bi and²²³Ra in bone after a few hours. For ²²⁴Ra based on animal data andextrapolation to adult human, it was found by two different models a²¹²Pb to ²²⁴Ra fraction of 0.88 and 1.0, i.e., almost complete retentionof daughters. These data indicates a high retention of the daughternuclides in bone for both ²²³Ra and ²²⁴Ra. For ²²⁴Ra a significantcontribution to soft tissue uptake of progeny would therefore likely befrom co-injected daughter nuclide. Thus it is imperative to developmethods to control daughter nuclides, at least ²¹²Pb, in ²²⁴Rainjectates.

This has been achieved by the new ²²⁴Ra solutions described herein.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a radiopharmaceuticalsolution comprising ²²⁴Ra and a complex capable of scavenging at least²¹²Pb.

In one embodiment of the present invention, the complex comprises one ormore compounds selected from the group consisting of acyclic chelators,cyclic chelators, cryptands, crown ethers, porphyrins or cyclic ornoncyclic polyphosphonates. DOTMP, EDTMP, bisphosphonate, pamidronateconjugated to DOTA or TCMC.

In another embodiment of the present invention, ²¹²Pb and/or ²¹²Bi iscomplexed by bone-seeking EDTMP.

In another embodiment of the present invention, the complexing agent isconjugated to a compound selected from the group consisting of amonoclonal, a polyclonal antibody, an antibody fragment, a syntheticprotein, peptide, vitamin or vitamin derivative.

In another embodiment of the present invention, the complexing agent isthe chelator TCMC conjugated to a compound selected from the groupconsisting of a monoclonal, a polyclonal antibody, am antibody fragment,a synthetic protein, peptide, vitamin or vitamin derivative.

One aspect of the present invention relates to a kit comprising a firstvial comprising a radiopharmaceutical solution of the present invention,and a second vial comprising a neutralizing solution to adjust pH and/orisotonicity of the radiopharmaceutical solution prior to administrationto a patient.

Another aspect of the present invention relates to a kit comprising afirst vial comprising a chelate labeled protein or peptide, a secondvial comprising a ²²⁴Ra solution.

A further aspect of the present invention relates to aradiopharmaceutical solution of the present invention for use as amedicament.

Yet another aspect of the present invention relates to aradiopharmaceutical solution of the present invention for use intreating skeletal disease.

In one embodiment of the present invention, the skeletal disease isselected from the group consisting of skeletal metastases from cancersto the breast, prostate, kidneys, lung, bone, or multiple myeloma, ornon-cancerous diseases causing undesired calcification includingankylosing spondylitis.

A further aspect of the present invention relates to a method oftreatment of malignant or non-malignant disease by administration of aradiopharmaceutical solution of the present invention to an individualin need thereof.

Another aspect of the present invention relates to a method forproviding a ²²⁴Ra solution comprising protein-complex or peptide-complexcomprising mixing of chelate labeled protein, e.g., a monoclonalantibody, or peptide with a solution comprising ²²⁴Ra.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the decay of ²²⁴Ra and daughters.

FIG. 2 shows the decay of ²²³Ra and daughters.

FIG. 3 A shows the biodistribution in nude mice of ²²⁴Ra and progeny²¹²Pb in a solution without chelator. FIG. 3 B shows biodistribution innude mice of ²²⁴Ra solution containing EDTMP as chelator for progeny²¹²Pb. FIG. 3 C shows biodistribution in nude mice of ²²⁴Ra solutioncontaining TCMC-trastuzumab (Herceptin) as chelator for progeny ²¹²Pb.

FIG. 4 shows Table 1. Main radiation properties from the ²²⁴Ra series.¹Average per ²²⁴Ra transformation due to branching. Only X-rays orgammas above 1% effective abundance are accounted for. It adds up to atotal effective energy of approximately 26.5 MeV of alpha of 0.7 MeV ofbeta per complete decay of ²²⁴Ra and daughters.

FIG. 5 shows Table 2. Ingrowth of ²¹²Pb from a pure 10 MBq ²²⁴Ra source.Changes in activity level. Start activity 10 MBq pure 224-Ra.

FIG. 6A shows: Table 3 A. Thin layer chromatographic (TLC) profiles of²¹²Pb in a solution of ²²⁴Ra in equilibrium with progeny nuclideswithout and with complexing agents. Part 1: Phosphonates and formulationbuffer (FB).

FIG. 6B shows: Table 3 B: Thin layer chromatographic (TLC) profiles of²¹²Pb in a solution of ²²⁴Ra in equilibrium with progeny nuclideswithout and with complexing agents. Part 2: Table 3 B shows TLC profilesfor ²¹²Pb in the presence of antibody-TCMC or DOTA conjugates andantibody without chelator.

FIG. 7 shows Table 4. Uptake ratios* for ²¹²Pb for bone vs. blood andbone vs. kidneys.

The present invention will now be described in more detail in thefollowing.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Some abbreviations used

-   -   DOTMP—1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylene        phosphonic acid)    -   EDTMP—ethylenediamine tetra(methylene phosphonic acid)        EDTA-ethylenediaminetetraacetic acid    -   p-SCN-Bn-DOTA—2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic        acid    -   DOTA-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid        and also used for        benzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid        conjugated to monoclonal antibody    -   p-SCN-Bn-TCMC—2-(4-isothiocyanotobenzyl)-1, 4, 7, 10-tetraaza-1,        4, 7, 10-tetra-(2-carbamonyl methyl)-cyclododecane    -   TCMC—1, 4, 7, 10-tetraaza-1, 4, 7, 10-tetra-(2-carbamonyl        methyl)-cyclododecane and also used for benzyl-1, 4, 7,        10-tetraaza-1, 4, 7, 10-tetra-(2-carbamonyl        methyl)-cyclododecane conjugated to monoclonal antibody    -   mAb—monoclonal antibody

The same abbreviations are in the following used for acids, salts orpartly or fully dissociated versions of the chelators.

It has been an unexpected finding that it is possible to stronglycomplex daughter nuclide in the presence of radium with complexingcompounds tested, EDTMP and monoclonal antibody conjugated to the TCMCand DOTA chelators, and at the same time keeping radium mainly as anuncomplexed cation fully targetable to bone.

A Radiopharmaceutical Solution

An object of the present invention is to provide a radiopharmaceuticalsolution comprising ²²⁴Ra and a complex capable of scavenging at least²¹²Pb.

In the present context is “scavenging” defined as at least 50% boundaccording to thin layer chromatography (TLC), centrifuge concentrationseparation or biodistribution profiles.

This means, as an example, at least 50% less blood uptake of ²¹²Pb witha small molecular chelator. With an antibody conjugated chelator, whereblood uptake is not a reliable indicator, at least 50% bound accordingto TLC analyses.

In one embodiment of the present invention is at least 60% bound.

In another embodiment of the present invention is at least 70% bound.

In another embodiment of the present invention is at least 80% bound.

In another embodiment of the present invention is at least 85% bound.

In another embodiment of the present invention is at least 90% bound.

The compound or compounds may also be capable of scavenging moreradionuclides than ²¹²Pb.

In one embodiment of the present invention, the complex comprises one ormore compounds selected from the group consisting of acyclic chelators,cyclic chelators, cryptands, crown ethers, porphyrins or cyclic ornoncyclic polyphosphonates. DOTMP, EDTMP, bisphosphonate, pamidronateconjugated to DOTA or TCMC.

In one embodiment of the present invention, the complex is at aconcentration of 1 ng/mL to 1 g/mL.

In another embodiment of the present invention, the complex is at aconcentration of 100 ng to 10 mg/mL

The complex can comprise one, two, three, four, five or more compounds.

In another embodiment of the present invention, ²¹²Pb and/or ²¹²Bi iscomplexed by bone-seeking EDTMP.

In one embodiment is the solution in a volume of 100 μL to 1000 mL, suchas 500 μL to 100 mL, 1 mL to 10 mL.

In one embodiment of the present invention is the radioactivity of thesolution 1 kBq to 1 GBq, such as 10 kBq to 100 MBq, such as 100 kBq to10 MBq.

In another embodiment of the present invention is the radioactivity ofthe solution 100 kBq to 100 MBq.

In another embodiment of the present invention, the complexing agent isconjugated to a compound selected from the group consisting of amonoclonal, a polyclonal antibody, an antibody fragment, a syntheticprotein, a peptide, a hormone or hormone derivative or a vitamin orvitamin derivative, e.g., biotin and folate.

In another embodiment of the present invention, the complexing agent isthe chelator TCMC conjugated to a compound selected from the groupconsisting of a monoclonal, a polyclonal antibody, am antibody fragment,a synthetic protein, a peptide, a hormone or hormone derivative or avitamin or a vitamin derivative.

For dosing purposes ²²⁴Ra solutions administered should have been storedfor some time, e.g., 1 day or more preferably at least two days, such as1-2 days or 1-3 days, to reach equilibrium between ²²⁴Ra and²¹²Pb/²¹²Bi. This will ensure ²¹²Pb to ²²⁴Ra activity ratios between0.83 and 1.14. This can, e.g., be accomplished by the manufacturer bysimply retaining the product for a day or so before shipment.

It is important to keep radium as mainly an uncomplexed, or weaklycomplexed cation as this ensures a maximum uptake in bone and bonemetastases and also ensures favorable excretion of eliminated productmainly through the intestines.

By adding complexing agent to a solution of radium the radioactivedaughter can be made bone- or tumor-seeking and increase the therapeuticpotential of the radium solution instead of being a health hazard. Itshould be a complexing agent that does not negatively affect thebone-seeking properties of radium, though. For example can EDTMPscavenge ²¹²Pb produced in the radium solution during transport andstorage between the production site and the hospitals whereby theproduct is going to be administered.

Although it is possible to reduce the susceptibility by addingradiolytic inhibitors, tumor targeting proteins or peptides are oftenmore susceptible to radiolysis and should probably be supplied in a kitformat whereby they are added a few hours to a few minutes beforeadministration of ²²⁴Ra solutions with relatively long shelf-lives.

It is known in the field that calixarenes and EDTA to some extent cancomplex radium and also complex lead and bismuth. However, in thecurrent work we found chelators that would leave radium mainlyuncomplexed or weakly complexed, as determined by in vivobiodistribution measurements, while being able to rapidly and withrelevant stability, complex the longest living daughter ²¹²Pb. Theselective complexation can be used to make at least lead bone- ortumor-seeking while maintaining the favorable properties of radium interms of treating sclerotic diseases, like skeletal metastases. The²¹²Pb complex that targets bone or tumor cells generates the alphaemitter ²¹²Bi from the decay of ²¹²Pb. Thus, the beta emitter ²¹²Pb isused as an indirect alpha source for irradiating the targeted cells ortissue. Other potential chelates which could be suitable for ²²⁴Radaughter nuclide scavenging besides TCMC and DOTA includes but are notlimited to phorphyrins, DTPA and DTPA derivatives and also carboxyllinked DOTA.

Lead-212 is by far the longest living of the progenies from ²²⁴Ra andthis is the most important to complex, as it is an in vivo generator forthe short lived alpha-emitter ²¹²Bi. If a ²¹²Pb-chelate is taken up inbone or in tumor cells ²¹²Bi will also likely be retained in the target.In a ²²⁴Ra solution in equilibrium with progenies there will be morethan 10 times of ²¹²Pb vs. ²¹²Bi atoms. Thus, the amount of radiationgenerated from the ²¹²Bi atoms in these solutions are modest andprobably not of a toxicologically importance compared with ²²⁴Ra and²¹²Bi decay series. The amount of ²¹²Bi s comparable to that of the²¹¹Pb which indirectly produces an alpha particle in the ²²³Ra seriesand this has not been of a significant problem for the registration andclinical use of ²²³Ra in equilibrium with progenies.

If, however, a high degree of chelation also of ²¹²Bi in an injectateshould be needed, it may at least in some instances be necessary to adda stabilizing agent like NaI or HI since bismuth in aqueous solutionstends to exist in a state less suitable for chelation.

When comparing with current approved alpha-pharmaceutical for treatmentof skeletal metastases, i.e., ²²³Ra, the novel solutions describedherein could give, in one of the embodiment, a product with improvedproperties for treatment of skeletal metastases since the daughternuclide can be made targetable to circulating cancer cells and, to someextent, also soft tissue metastases. This may prevent recurrence fromcancer recolonization of the skeleton due to CTC's.

Another aspect is that the shorter half life of ²²⁴Ra vs. ²²³Ra mayactually be of some benefit as the radium is embedded in the bonematrix. Because of the high density of the bone mineral the range ofalpha-particles is strongly reduced in bone vs. soft tissues. Especiallyin rapid mineralizing areas like osseous cancer metastases, theembedment process may be of significance when using a volume-seekingalpha-pharmaceutical.

Therefore, ²²⁴Ra could improve the tumor dose since, on average, it willbe less embedded at the time of decay.

Diseases by which the novel ²²⁴Ra solutions may be used include but arenot limited to primary and metastatic cancers, autoimmune diseases andartherioschlerosis. The product may be administered intravenously orlocally, including intraperitoneally, or in limb perfusion settings.

The chelators used in the novel solutions may be acyclic as well ascyclic chelators and cryptands, crown ethers, porphyrins or cyclic- ornoncyclic polyphosphonates including DOTMP and EDTMP. Also abisphosphonate, e.g., pamidronate, conjugated to DOTA, TCMC or similarmay be used as scavenger in the ²²⁴Ra solution.

One may argue that the amount of ²¹²Pb in therapeutic ²²⁴Ra solution maybe moderate to modest (i.e., at equilibrium about 1.1 times that of²²⁴Ra). If one assume similar dosing of ²²⁴Ra as is done with ²²³Ra inpatients but correct for the half life difference, roughly 150 kBq perkg of bodyweight would be the administered dose.

At equilibrium this would translate into a ²¹²Pb-antibody conjugatedosage of 11.5 MBq in 5 litres of blood in a 70 kg patient (if ²¹²Pb isquantitatively chelated). The number of circulating tumor cells istypically less that 10 cells per ml thus in 5 l blood there are lessthan 50 000 tumor cell in total. If only 1 in 100 000 of the injected²¹²Pb-antibody conjugate molecules binds to the tumors cells this wouldmean at least 0.0023 Bq per cell, equivalent to approximately 127 ²¹²Pbatoms bound per cell, which would be highly destructive as it has beenreported that a mean of 25 cell bound ²¹²Pb per cell would kill 90% of acell population.

Previously it was suggested to combine radium and phosphonates and morepreferentially bisphosphonates in the treatment of skeletal metastasesof cancer. However, it was suggested to use the two compounds separatedfrom each other as it was preferred to inject at different time points.The application of phosphonates was not indicated for radionuclidecomplexation. The main purpose was to use pharmacologically activeamounts of phosphonates as a secondary bone treatment to radium. Also itwas preferred to use a non-complexing bisphosphonates, thus this teachesaway from using EDTMP or similar as additive to radium solutions for thecomplexation of daughter nuclides. At the time it was common knowledgethat EDTMP could complex alkaline earth metals, as it was known in thefield that ¹⁵³Sm-EDTMP can cause complexing of calcium in blood andcause hypocalcemia.

However, in the current work we have shown that when modest amounts ofEDTMP is used, it is possible to complex ²¹²Bi and ²¹²Pb withoutsignificantly reducing the bone-seeking properties of ²²⁴Ra.

The current report is the first time the addition of a complexingphosphonate to a radium solution has been presented. It shows that it ispossible to obtain selective complexation of daughter nuclide withoutsignificantly alter the bone targeting properties of radium. This isimportant since although complexing phosphonates are bone-seekers,radium shows an even higher bone targeting ability than thephosphonates. It is therefore highly advantageous that the labeling ofdaughter nuclides does not cause reduced skeletal uptake of the radium.

As for bone targeting with phosphonates it is known in the field thatradionuclides like ¹⁷⁷Lu, ¹⁵³Sm, ²²⁷Th, and ²²⁵Ac complexed withphosphonates can target bone. In a previous report it was also shownthat the radionuclides ²¹²Pb and ²¹²Bi could be complexed with EDTMP andDOTMP. However the labeling was performed with high pH and also neededto be purified by ion exchanger after labeling. Thus this teaches awayfrom using in situ labeling in the presence of radium, without affectingradium, and without purification as is shown in the current application.Thus, we hereby present a novel way of using EDTMP as a scavenger fordaughter nuclides in ²²⁴Ra solutions which (1) improve the bone dose perunit ²²⁴Ra administered, and (2) strongly reduce uptake of ²¹²Pb inhematopoietic cells and tissues in effect causing better target tonon-target radiation ratios.

The solutions used for ²²⁴Ra and the daughter nuclide complexation maycontain radiolytic inhibitors and other modificators suitable for amedical injectate known in the field.

The solution can also be a pharmaceutical composition.

Usually is an important element of a pharmaceutical composition a buffersolution, which to a substantial degree maintain the chemical integrityof the radioimmunoconjugate and is being physiologically acceptable forinfusion into patients.

In one embodiment of the present invention the pharmaceuticalcomposition comprises one or more pharmaceutically acceptable carriersand/or adjuvants.

Acceptable pharmaceutical carriers include but are not limited tonon-toxic buffers, fillers, isotonic solutions, etc. More specifically,the pharmaceutical carrier can be but are not limited to normal saline(0.9%), half-normal saline, Ringer's lactate, 5% Dextrose, 3.3%Dextrose/0.3% Saline. The physiologically acceptable carrier can containan anti-radiolytic stabilizer, e.g., ascorbic acid, which protect theintegrity of the radiopharmaceutical during storage and shipment.

Kits

The solution should be made physiologically suitable for injectionseither at a centralized production site or be made up by a kit system oftypically 2-4 vials whereby being physiologically suitable for injectionafter combination of the kit vials.

One aspect of the present invention relates to a kit comprising a firstvial comprising a radiopharmaceutical solution of the present invention,and a second vial comprising a neutralizing solution to adjust pH and/orisotonicity of the radiopharmaceutical solution prior to administrationto a patient.

Another aspect of the present invention relates to a kit comprising afirst vial comprising a chelate conjugated to a protein or peptide ormixtures of proteins or peptides, and a second vial comprising a ²²⁴Rasolution.

Since the decay series of ²²⁴Ra includes a radon daughter which maydiffuse into the air, vials containing the products must be well sealedto prevent escape of ²²⁰Rn.

Because of the highly localized nature of alpha-irradiation, radiolysismust be considered as a potential problem and the radiopharmaceuticalmust be designed to minimize this. According to the knowledge in thefield radiolabeled antibodies are sensitive to radiolysis and thereforea kit system may be advantageous for ²²⁴Ra solutions which are to becombined with chelator conjugated antibodies for scavenging ²¹²Pb and or²¹²Bi.

For a monoclonal antibody it is usually advisable be keep the self doseof the alpha particle producing radiopharmaceutical solution below 0.5kGy to avoid reduced binding properties due to radiolysis. Thus, a kitsystem whereby chelator conjugated antibody is added to the ²²⁴Ra(including daughters) solution a few hours to 10 minutes beforeinjection is advised for concentrated solutions intended for remoteshipping.

In one embodiment of the present invention, the chelator conjugatedantibody is added to the ²²⁴Ra (including daughters) solution 30 min to1 hour before injection.

In one embodiment of the present invention, the chelator conjugatedantibody is added to the ²²⁴Ra (including daughters) solution 1 min to20 min before injection.

In one embodiment of the present invention, the chelator conjugatedantibody is added to the ²²⁴Ra (including daughters) solution 1 min to10 min before injection.

A kit with a chelate labeled protein or peptide in one vial and a ²²⁴Rasolution in another vial whereby the content of the two are mixed a fewhours to 30 minutes before administration to a patient as to bind ²¹²Pband or ²¹²Bi to the chelate.

In one embodiment of the present invention, the content of the two aremixed 30 min to 1 hour before injection.

In one embodiment of the present invention, the content of the two aremixed 1 min to 20 min before injection.

In embodiment of the present invention, the content of the two are mixed1 min to 10 min before injection.

Optionally, a third vial containing a liquid used for dilution andisotonicity adjustment before administration of the radiopharmaceuticalsolution could be used. This third vial may contain EDTMP which couldchelate ²¹²Bi, if needed.

Medical Uses

The described novel methods and solutions have thereby solved a majorproblem for the application of ²²⁴Ra in nuclear medicine.

Thus two types of diseases can be treated with the new formulations:

-   -   1. Pure bone related, or sclerotic diseases with radium and        daughter nuclide complexed by a phosphonate.    -   2. Bone-related disease with soft tissue or circulating target        cells components by radium and daughter nuclide complexed to        chelate-monoclonal antibody conjugate or similar protein or        peptide conjugates. A special embodiment of this would be when        ²¹²Pb is conjugated to a protein or peptide chelate and the        ²¹²Bi is complexed by a bone seeker, e.g., EDTMP.

Proteins or peptides may be used with the current invention includingthose targeting osteosarcoma, lung cancer, breast cancer, prostatecancer, kidney cancer, thyroid cancer.

Thus, a further aspect of the present invention relates to aradiopharmaceutical solution of the present invention for use as amedicament.

Yet another aspect of the present invention relates to aradiopharmaceutical solution of the present invention for use intreating skeletal disease.

In one embodiment of the present invention, the skeletal disease isselected from the group consisting of skeletal metastases from cancersto the breast, prostate, kidneys, lung, bone, or multiple myeloma, ornon-cancerous diseases causing undesired calcification includingankylosing spondylitis.

A further aspect of the present invention relates to a method oftreatment of malignant or non-malignant disease by administration of aradiopharmaceutical solution of the present invention to an individualin need thereof.

Method of Production

Herein are described novel methods possible to produce a ²²⁴Ra solutionsuitable for treating skeletal diseases including primary or metastaticcancer, using centralized production and up to several days of shipmentand or storage before administration to patients.

Moreover, such a solution can give higher initial tumor radiation dosecompared to a pure ²²⁴Ra solution since the daughter products will giveadditional dose to tumor when they are made bone-seeking or tumorseeking by the aforementioned chelating complexes. This could make ²²⁴Rasolutions more potent in cancer therapy since the mother nuclide cantarget the bone disease while the longest lived daughter nuclide can byan added complex be made to seek out and destroy circulating cancercells in the blood, alternatively be made a purer bone-seeker byphosphonate complexation.

The novel procedures and methods presented here allows the productionand shipment of ²²⁴Ra with longer shelf life of days or even up to aweek or longer since the “problematic” daughter nuclide can be scavengedby tumor-seeking chelates and actually enhance the therapeuticproperties of the ²²⁴Ra solutions.

This finding is important since ²²⁴Ra has been considered less usefulfor e.g., cancer therapy against skeletal metastases because of daughterproducts with substantial half-lives, in particular ²¹²Pb, which will bepresent in significant quantities a few hours after the production of apure ²²⁴Ra solution.

That is made possible by the novel pharmaceutical solutions describedhere were the ²²⁴Ra targets bone and bone metastases while ²¹²Pb can bemade to target circulating tumor cells depending on the chelate-antibodyconjugate used. The pharmaceutical solution could be “taylor made”according to the primary tumor from which the skeletal metastasesoriginate. There exist several antibodies with selectivity for differentantigens expressed in e.g., prostate-, breast-, lung-, bone-, kidney-,thyroid- and multiple myeloma cancers.

Thus, another aspect of the present invention relates to a method forproviding a ²²⁴Ra solution comprising protein-complex or peptide-complexcomprising mixing of chelate labeled protein or peptide with a solutioncomprising ²²⁴Ra.

Thus, the novel invention described herein can be used either as a purebone-seeker when combined with EDTMP or similar, or if patients havemeasurable CTC's or is suspected of such, can be used as a combinationof treatment of bone metastases and CTC's or soft tissue metastases,thereby adding a new dimension to alpha pharmaceuticals against bonerelated disease, i.e., an additional prophylactic activity preventingsettling of viable CTC's in the skeleton or soft tissues.

The novel and surprising findings are as follows:

-   -   1. Radium solutions can be conditioned with complexing        phosphonates or complex conjugated antibodies without causing        complexation of radium and causing reduced bone uptake of radium        while complexing ²¹²Pb and/or ²¹²Bi in situ without a need for        purification before use.    -   2. Daughter nuclides produced during shipment and storage can be        complexed effectively in situ yielding a radium solution with        improved daughter nuclide properties.

The ²²⁴Ra solution can be stored or shipped for several days and stillthe major fraction of the ²¹²Pb generated can be complexed, i.e., atleast 60%, more favorable at least 90%, and even more favorable 95-100%,depending on the amount of complexing agent added.

This is important since it gives a radiopharmaceutical with betteroverall target to non-target ratios compared to existing radiumformulations as shown in following examples and allows the use of ²²⁴Rasolutions that has been shipped and stored for several days. Indeed itteaches away from using freshly prepared ²²⁴Ra solutions with only fewhours shelf life as in the method of Altmann Terapie, since it may beadvantageous with the new method that ²¹²Pb has reached equilibrium orclose to equilibrium, i.e., at least 1 day or more, to obtain areproducible and defined ratio between ²²⁴Ra and ²¹²Pb in theadministered radiopharmaceutical solution.

We have also confirmed that other complexes than phosphonates can belabeled with daughter nuclide in situ in radium solutions withoutsignificantly affecting the radium, e.g., chelate-antibody conjugateswere added to radium solution and showed a relevant labeling of daughternuclide with a significant preservation of the chemical integrity ofradium, thus it is possible to have a radium solution whereby one ormore of the daughter nuclides is complexed to tumor-seeking moleculesyielding a pharmaceutical solution with bi-specific targetingproperties, e.g., radium targeting the skeletal disease and a chelatecomplex with daughter nuclides targeting a tumor cell antigen oncirculating tumor cells etc. As a special embodiment one daughternuclide can be complexed to a monoclonal antibody and the other to aphosphonate.

In a special embodiment of the current invention is a solution withradium containing TCMC conjugated to a protein or a peptide,preferentially a monoclonal antibody, whereby the antibody is targetingan antigen on breast-, prostate-, lung-, kidney-, bone- or multiplemyeloma cancer cells. If left for a few minutes to a few days, the ²¹²Pbgenerated will mainly bind to the TCMC-antibody conjugate. When such asolution is injected into a cancer patient, ²²⁴Ra will protect the bonefrom destruction by killing tumor cells at the bone surface and in theskeleton and the ²¹²Pb labeled antibody will kill circulating cells outof range from the radium radiation. Thus, what was before a problem offree daughter nuclides with undesired biodistribution is made into anadvantage for the ²²⁴Ra series by the use of the current invention. Itis particularly attractive to use an internalizing antigen as targetsince ²¹²Pb-conjugates are especially effective as in vivo generator foralpha-emitters under such conditions whereby the alpha emitting ²¹²Bidaughter is enclosed in the target cells (Boudousq et al., 2013).

In one embodiment the radiopharmaceutical product is shipped ready touse, e.g., in a vial with a septum for withdrawal of the solution with asyringe or even as a prefilled syringe ready to use. In a secondembodiment the product may be shipped in a kit format consisting of avial with ²²⁴Ra solution, a second vial with a solution of a chelatorand optionally a third solution with a formulation buffer for theadjustment of concentration and/or pH etc. The chelator, whereby it is aphosphonate, chelator-antibody conjugate or similar, is added to the Rasolution and mixed for a few minutes to a few hours most preferable from5-60 minutes, before the product is, if needed added a formulationbuffer, and administered to the patient.

EXAMPLES Example 1 Calculation of the ²¹²Pb Daughter Nuclide Level from²²⁴Ra Decay at Various Time Points

Background. The ²¹²Pb produced after preparation of a pure ²²⁴Raradiopharmaceutical may be a problem since it has different andundesirable properties compared with the mother nuclide. E.g., it isknown that radium can target bone and bone metastases, but the leadprogeny has undesired accumulation in hematopoietic cells and tissuesand in the kidneys.

Method: The ingrowth of ²¹²Pb from a pure ²²⁴Ra source were calculatedusing a universal activity calculator.

Results: Table 2 shows the amount of ²¹²Pb at various time points afterthe production of a pure ²²⁴Ra pharmaceutical solution and storage in agas tight container.

The data shows that significant amount of daughter nuclide is presentwithin a relatively short time frame, complicating a potentialcentralized production and supply of ²²⁴Ra based radiopharmaceuticals.It is noteworthy though that the ratio of ²¹²Pb to ²²⁴Ra in the solutionreaches 1 after 36 hours and thereafter gradually increases to about 1.1of which it stay for the rest of the time until complete decay.

Example 2 Preparation of Radionuclides and Counting of RadioactiveSamples

In the following, all work with the concentrated radioactivepreparations including evaporation of solvent etc was performed in aglove-box. A source of ²²⁸Th in 1 M HNO₃ was acquired from a commercialsupplier. Ac-resin was obtained from Eichrom Technologies LLC (Lisle,Ill., USA) in the form of a pre-packed cartridge.

To use smaller volume of solvent, about thirty percent of the materialsin a cartridge (Cartridge 1) was extracted and repacked in a smallercolumn (Cartridge 2) made by a 1 ml filtration column (Isolute SPE,Biotage AB, Uppsala, Sweden). A slurry representing 20% of the originalcartridge content was used for immobilizing of ²²⁸Th in 500 mikroliter 1M HNO3 which was added 500 microliter of 1 M HCl and incubated byshaking the vial (4 ml vial, E-C sample, Wheaton, Millville, N.J., USA)for at least 4 hours. Cartridge 2 was added a small amount (about 0.1ml) of the Ac-resin. Thereafter, the slurry was added to cartridge 2using the prefilled material as a catcher layer. Radium could be elutedfrom the Cartridge 2 in 2 ml of 1 M HCl. The 2 ml radium solution wasevaporated to dryness, using a heater block and flushing the vial withN2 gas through a Teflon tube inlet and outlet in the rubber/Teflonseptum on the vial and by leading the acid vapor into a beaker ofsaturated NaOH by a stream of N₂-gas.

The residue was resolved in 0.5 ml 1 M HNO₃ and loaded to a cartridge 3consisting of a 1 ml Isolute column packed with about 250 mg Dowex anionexchanger. Cartridge 3 was washed with 7 ml 1 M HNO₃, which removed²¹²Pb, and finally with 3-4 ml 8 M HNO₃ to elute ²²⁴Ra. The ²²⁴Ra eluatewas evaporated to dryness, using the heater block and a flow of N₂-gas,and the residue could be dissolved in 0.1 M HCl. Typically, more than70% of the ²²⁴Ra present in the ²²⁸Th source could be extracted andpurified using the described methods.

Radioactive samples were counted on a Cobra II Autogamma counter(Packard Instruments, Downer Grove, Ill., USA). During extraction of²²⁴Ra from the ²²⁸Th source, a CRC-25R dose calibrator (Capintec Inc.,Ramsey, N.J., USA) was used.

To determine distribution of ²²⁴Ra, ²¹²Pb and ²¹²Bi in real time insamples, a liquid nitrogen cooled HPGe detector (GWC6021, CanberraIndustries, Meriden Conn., USA) was used. This was combined with a DSA1000 digital signal analyzer and the Genie 2000 software (Canberra).

Example 3 Determining Net Count Rate for ²¹²Pb in a ²¹²Pb/²²⁴Ra MixtureBefore Radioactive Equilibrium has been Reached

After more than 3 days, i.e., “equilibrium” a sample will for practicalpurposes have 1.1 times ²¹²Pb vs ²²⁴Ra.

Regardless of whether ²¹²Pb is higher or lower than equilibrium it canbe assumed that this is reached after 3 days since surplus ²¹²Pb isreduced by 99% and the ingrowth of ²¹²Pb from ²²⁴Ra is practicallycomplete vs. “equilibrium”.

Using the Cobra II Autogamma counter with a counting window setting from70-80 KeV gives mainly the ²¹²Pb with very little contribution fromother radionuclides in the ²²⁴Ra series. Radium-224 must be indirectlycounted when the initial ²¹²Pb has vanished and equilibrium between²²⁴Ra and ²¹²Pb has been reached (after approximately 3 days). Thisindirect counting requires the sample to be stored in a relatively gastight containers as otherwise the ²²⁰Rn may escape preventing theradionuclide equilibrium of 1.1 between ²¹²Pb and ²²⁴Ra to be reached.

Since sampling and counting may be separated by some time, the net countrate for ²¹²Pb can be adjusted for decay to determine the net ²¹²Pbcount rate at the time of sampling.

Example 4 Thin Layer Chromatography Analyses

Thin layer chromatography (TLC) was performed using chromatographystrips (model #150-772, Biodex Medical Systems Inc, Shirley, N.Y., USA).A small beaker with about 0.5 ml of 0.9% NaCl was used to place stripswith a sample spot in. To the strip was typically added 1-4 μl of sampleat approximately 10% above the bottom of the strip. After the solventfront had moved to about 20% from the top of the strip, the strip wascut in half and each half was placed in a 5 ml test tube for counting.In this system radiolabeled antibody and free radionuclide does notmigrate from the bottom half while radionuclide complexed with EDTAmigrates to the upper half. A formulation buffer (FB) consisting of 7.5%human serum albumin and 5 mM EDTA in DPBS and adjusted to approximatelypH 7 with NaOH was mixed with the antibody conjugates in ratio 2:1 forat least 5 minutes before application to the strips to determine freeradionuclide.

The analysis of the EDTMP was done without the FB (Table 3 A). Thelabeling of radionuclide with EDTMP was measured by the amount ofmigration to the upper half of strips. The DOTMP migrated poorly in thissystem and therefore these solutions had to be treated with FB tomeasure free radionuclide at the upper half of the strip (Table 3 A).

Example 5 In Situ Chelation of ²¹²Pb in ²²⁴Ra Solutions

Initially ²²⁴Ra solutions in 0.1 M HCl was neutralized with 1 M NaOH andthe EDTMP was added to the solution to obtain a pH slightly belowneutral. In later experiments 10:1 ratio of ²²⁴Ra in 0.1 M HCl and 5 Mammonium acetate was used before addition of the chelators, resulting ina pH range of 5.5-7 for the reactions. Reaction times of 30 minutes toseveral days, at room temperature, were tested for EDTMP with goodlabeling yield (typically above 90% according to TLC) whenconcentrations of about 4-8 mg/ml EDTMP in reaction solutions were used.Thus, EDTMP seems to be a good scavenger for ²¹²Pb in situ in ²²⁴Rasolutions. As for DOTMP the labeling was less efficient with about 70%labeling for 7 mg/ml at room temperature and about 1 h of reaction time.The labeling yield of DOTMP may be improved by adjusting chelatorconcentration or reaction time etc. It should be noted that subsequentexperiments using EDTMP and ammonium acetate buffers showed a goodlabeling with ²¹²Pb also in radium solutions of pH 5.5 to 7 asdetermined by thin layer chromatography (Table 3 A).

Long term scavenging was tested by leaving a ²²⁴Ra solution with EDTMP(approximately 6 mg/ml) buffered to about pH 6 with ammonium acetate,for 7 days at room temperature. Analysis of the ²¹²Pb distributionprofile was performed by use of TLC as described. Results: At least 93%of the activity was found in the upper half of the TLC-stripcorresponding to EDTMP associated activity after 7 days. In conclusion,it is possible to effectively chelate ²¹²Pb generated in situ in ²²⁴Rasolution by EDTMP. Thus, it is possible to prepare ready to use ²²⁴Rasolutions with a bone-seeking chelator that scavenge ²¹²Pb in situthereby obtaining ²²⁴Ra solution with improved shelf life for use as abone targeting radiopharmaceutical.

As an alternative, a labeling kit may be used whereby EDTMP is added toa several days old ²²⁴Ra solutions a few minutes to a few hours beforeadministration. Such a labeling kit will also allow centralizedproduction of ²²⁴Ra as the kit can be very simple to use for ²²⁴Rasolutions several days to more than a week after the production date for²²⁴Ra.

In an experiment with an 8 day old ²²⁴Ra solution in 0.1 M HCl and 0.5 Mammonium acetate, EDTMP to a concentration of about 7 mg/ml was added.After 10 minutes and 1 hours standing at room temperature, TLC analysesshowed 91% and 93%, respectively, of the ²¹²Pb was associated with theEDTMP.

EDTMP solutions up to 4 months old were tested and found to befunctional, thus EDTMP seems well suited to be used in a kit format.

Example 6 Biodistribution in Mice of ²²⁴Ra with Significant Amounts of²¹²Pb with and without EDTMP

The main objective was to study the biodistribution of the injectedradionuclides with or without EDTMP. An EDTMP containing- and a salinecontrol solution, respectively, of ²²⁴Ra in equilibrium with daughterradionuclides were used.

Materials and methods: Animal experiments were performed according toEuropean regulations for animals used for scientific purposes. Nude micewere fully grown and were at an age of more than 6 months. A 3 day old0.1 M solution containing ²²⁴Ra was split in two. One fraction was addedEDMP and 1 M NaOH to adjust the pH to approximately 8, to a finalconcentration of 5 mg EDTMP per ml (solution A). The other fraction wasadjusted by 1 M NaOH to approximately pH 7 (solution B). Each solutionwas sterile filtered through a 13 mm 0.2 μm Acrodisc syringe filter(Pall Life Science, Port Washington, N.Y., USA) with Supor membrane.Thereafter 100 μl with approximately 20 kBq of ²²⁴Ra were administeredby tail vein injection into each mouse.

Results: As shown in FIGS. 3 A and B there was a significant improvementin ²¹²Pb distribution when EDTMP was added. The soft tissue and blooduptake was significantly reduced while the bone targeting was similar asfree ²¹²Pb. Thus, the bone to soft tissue ratios were greatly improvedfor ²¹²Pb by adding EDTMP to the ²²⁴Ra solution. There is no significantchange in the ²²⁴Ra distribution when adding EDTMP as shown in FIGS. 3 Aand B.

Conclusion: addition of EDTMP to ²²⁴Ra solutions improves ²¹²Pbbiodistribution with no significant changes to the ²²⁴Rabiodistribution.

Example 7 Labeling of Chelator-Conjugated Antibody with ²¹²Pb In Situ in²²⁴Ra Solution

Background: It is advantageous from a logistic perspective thatradiopharmaceutical can be produced in a centralized production unit andshipped to the end user. The ²¹²Pb generated should be scavenged by thechelator to minimize injection of free ²¹²Pb when ²²⁴Ra is used.

Methods: ²²⁴Ra was produced and purified as described in example 2.TCMC- and DOTA-labeled monoclonal antibodies were prepared by usingantibodies purified with centrifuge concentrator (Vivaspin 4 or 20, 50000 MWCO, Sartorius Stedim, Goettingen, Germany) and added 150 mMcarbonate buffer, pH 8.5-9. The antibody had a concentration oftypically 20-30 mg/ml and was added p-SCN-Bn-TCMC or p-SCN-Bn-DOTA(Macrocyclics Inc, Dallas, Tx, USA) using antibody to chelator ratios of1:9 or 1:5, respectively. After at least two hours incubation at roomtemperature the reaction was terminated by adding 0.1 M glycine incarbonate buffer (pH approximately 8.5) and further incubation for 10minutes before purification and buffer exchange into 0.9% NaCl usingcentrifuge concentrator (Vivaspin). Chelator-antibody concentrations of15-35 mg/ml in 0.9% NaCl were used as stock solutions.

To a 2 ml eppendorf tube was added typically 40 μl ²²⁴Ra in 0.1 M HCl, 5μl of 5 M ammonium acetate, 5-10 μl, (15-30 mg/ml) TCMC- or DOTA-labeledantibody in 0.9% sodium chloride. This method was tested for 4 differentantibody conjugates including those of trastuzumab (Herceptin),rituximab, cetuximab, and 01-3 murine monoclonal antibody. The pH wasdetermined to be in the range of 5.4-6.0 by applying 1 μl on a pH paper(No 1.09564.0003 and 1.09556.003 from Merck KGaA, Darmstadt, Germany)and reading the colour. The reaction was performed at room temperature.

In some of the experiments a control solution with the same ingredientsexcept for that the antibody did not contain TCMC or DOTA was reacted inparallel using the same conditions. After 30 and 100 minutes 5 μl waswithdrawn and mixed with 10 μl of a formulation buffer (FB) consistingof 7.5% human serum albumin and 5 mM EDTA in DPBS. After at least 10minutes, 1-4 μl of the product/FB mixture was withdrawn and placed on athin layer chromatography strip (Biodex). The same procedure wasperformed with the control solution. The strips were eluted in 0.9% NaClsolution and when the solvent front reached almost to the top, the stripwas removed, cut in half and the bottom and top part was countedseparately on a Cobra II gamma counter (as previously described).

Results: The thin layer profiles are presented in Table 3 B. Typicallymore than 90% of the activity was found at the bottom half of the thinlayer strip for the TCMC- and DOTA-antibody conjugate. In the control,typically more than 97% of the activity was found in the top half of thestrip. This shows that both TCMC- and DOTA-conjugated antibodies can beefficient scavengers for ²¹²Pb in ²²⁴Ra solution. Using a centrifugefiltering unit with a cut-off of 37 kDa it was also shown that ²²⁴Ra inthe reaction solution could be separated effectively from the TCMC- orDOTA conjugates by washing with 0.9% NaCl solution. By using 2 ml andconcentrate to 0.25 ml>85% of the radium was removed from the²¹²Pb-DOTA-antibody conjugate, thus it shows that TCMC- andDOTA-antibody conjugates can scavenge ²¹²Pb without significantlycomplexing ²²⁴Ra. In conclusion, it is possible to use TCMC orDOTA-antibody conjugate to scavenge ²¹²Pb in ²²⁴Ra solution thus,enabling the production of a pharmaceutical solution with dual targetingproperties, i.e., a bone seeking ²²⁴Ra and an antigen seeking ²¹²Pbconjugate.

In a follow-up experiment a ²²⁴Ra solution, added TCMC-labeled chOI-3(chimeric OI-3) monoclonal antibody conjugate (to about 1.5 mg/ml),buffered to about pH 5.5 with ammonium acetate and kept for 7 days atroom temperature. Thereafter samples were withdrawn and mixed 1:2 withformulation buffer (as described) and after 5 minutes or more applied onTLC strips as described. It was found that on average 95.6% was retainedwith the protein (lower half of the strip). In conclusion ²¹²Pb isscavenged effectively in situ in ²²⁴Ra solution over several days byTCMC-labeled antibody. Thus, it shows that centralized productionrequiring storage and shipment for several days is possible for ²²⁴Rasolutions with chelate-antibody conjugate.

Example 8 Cell Binding Experiment with Radiolabeled Monoclonal Antibodyin Mixture with ²²⁴Ra

The human osteosarcoma cell line OHS expresses Her-2 (relatively weak)and MUC-18 (moderate). They were therefore used for evaluating cellbinding fraction of chelator conjugated trastuzumab and chOI-3antibodies against Her-2 and MUC-18, respectively. Radium-224 wasdissolved in 0.1 M HCl and left for two days to reach equilibrium with²¹²Pb and ²¹²Bi. To adjust pH 12 μl 5 M ammonium acetate in metal freewater was added to 100 μl of ²²⁴Ra in 0.1 M HCl and thereafter added 200μg of TCMC-labeled trastuzumab. After 30 minutes thin layerchromatography confirmed that more than 90% of the ²¹²Pb was scavengedby the chelator. The reaction mixture was sterile filtered using a 13 mmsyringe filter and the product tested for cell binding usingapproximately 10 million cells in 0.2 ml of DPBS with 0.5% BSA. Cellswere either blocked by incubation with 20 μg of the same antibody for 15minutes (to measure non specific binding) or left unblocked beforeadding reaction solution with about 10 ng of chelate-antibody conjugatemixed with radionuclide to each tube. After 1 hour of incubation, tubeswere counted on the Cobra II gamma counter to determine added activity.Thereafter the cells were washed three times with 0.5 ml DPBS/0.5% BSAby whirlmixing, centrifugation and removal of supernatant and then thecell bound activity in the tubes were measured. The percent of cellbound antibody conjugate was determined as bound after wash divided bythe added activity times 100. Results: When corrected for decay andradiochemical purity and the non-specific binding was subtracted, it wasfound that 64.3-72.2% of the ²¹²Pb-labeled antibodies were boundspecifically to the cells. In this one point assay this indicatesappropriate targeting properties of the ²¹²Pb-antibody conjugateproduced in situ in ²²⁴Ra solutions. Conclusion: It is shown that²¹²Pb-labeled conjugates with relevant tumor targeting properties can beproduced in situ in ²²⁴Ra solutions.

Example 9 Biodistribution in Mice of ²²⁴Ra/²¹²Pb with TCMC-LabeledMonoclonal Antibody

Background: To study whether ²²⁴Ra/²¹²Pb solutions could be used forpreparing skeletal targeted and tumor cell targeted co-therapeutics.

Materials and Methods: A one day old ²²⁴Ra solution as described inexample 6 was added NaOH, 5 M ammonium acetate and TCMC-labeledTrastuzumab in the same way as in example 7 and stored over night. Thesolution was added metal free water in a 1:1 ratio, sterile filteredusing a 13 mm 0.2 μm Acrodisc syringe filter (Pall Life Science, PortWashington, N.Y., USA) with Supor membrane. The cell binding ability ofthe ²¹²Pb-labeled antibody component in the ²²⁴Ra solution was verifiedas in example 8. Animal experiments were performed according to Europeanregulations for animals used for scientific purposes. Animals wereeuthanized and blood drawn from the heart before they were dissected.Urine, blood and tissue samples were placed in 5 ml tubes. The weight ofthe tubes were measured before and after addition of samples todetermine exact sample weight. The radioactivity content were measuredin the Cobra gamma counter. Samples were counted shortly after thedissection and again after 3-4 days when radioactive equilibrium hadbeen reached to determine ²¹²Pb and ²²⁴Ra content, respectively.

Results: The biodistribution profiles are shown in FIG. 3 C. The ²¹²Pbshowed a distribution profile as expected for a radiolabeled antibody,i.e., high activity in blood and blood rich tissues and low activity infemur and scull. Compared with free ²¹²Pb (FIG. 3 A), theTCMC-trastuzumab (Herceptin) conjugated ²¹²Pb had significantly lessuptake in femur and scull while the activity level in blood and bloodrich organs were higher. It should be noted that the quality of thedistribution is different in blood for free ²¹²Pb and²¹²Pb-TCMC-herceptin as the latter is circulating with a slow bloodclearance but not taken up in the blood cells as with the free ²¹²Pb.The ²²⁴Ra showed high uptake in femur and scull and low uptake in bloodand was very similar to that found in the biodistribution of chelatorfree radium solution (FIG. 3 A).

In Table 4 the uptake ratios for bone vs blood and bone vs. kidney for²¹²Pb, ²¹²PB-EDTMP and ²¹²Pb-TCMC-trastuzumab in ²²⁴Ra solutions arepresented. The ratios show improved bone to blood and bone to kidneyratios for ²¹²Pb-EDTMP compared with non-complexed ²¹²Pb. For²¹²Pb-TCMC-herceptin, however, the bone to blood ratios were lower thanfor free ²¹²Pb. This is expected since macromolecular sized monoclonalantibodies clear slowly from the blood compared with most smallmolecular weight compounds. This may be an advantage when targetingcirculating tumor cells as the increased residence time in bloodenhances the probability of binding to target cells in circulation. Itshould also be noted that for short radiation range alpha particles theeffect of cellular bound radionuclide may be much stronger than that ofcirculating radionuclide because of the short proximity to the DNA forcell associated-vs. the freely circulating radionuclide.

In conclusion: Chelator labeled monoclonal antibody can effectivelyscavenge ²¹²Pb without reducing the bone seeking properties of ²²⁴Ra inradiopharmaceutical solutions containing the two radionuclides. Thus, itshows the possibility of obtaining dual targeting properties of²²⁴Ra/²¹²Pb by adding complexing agents to the solutions, therebystrengthening the therapeutic potential of the radiopharmaceuticalsolution and reducing possible undesired uptake of ²¹²Pb inhematopoietic cells and tissues while upholding the bone seekingproperties of ²²⁴Ra.

Example 10 A Kit System for Avoiding Radiolysis

Because of the highly localized nature of alpha-irradiation radiolysismust be considered as a potential problem and the radiopharmaceuticalmust be designed to minimize this. According to the knowledge in thefield radiolabeled antibodies are sensitive to radiolysis and thereforea kit system may be advantageous for ²²⁴Ra solutions which are to becombined with chelator conjugated antibodies for scavenging ²¹²Pb. When²²⁴Ra is in equilibrium with progenies it produces approximately 28 MeVper decay of the complete series. Thus, a solution of 1 MBq/ml containsN=A/λ=10⁶ s-1/(0.693/[3.64×24×3600 s])=4.53×10¹¹ atoms of ²²⁴Ra where Nis the number of atoms and A is activity in Bq and λ is the decaysconstant which is equal to ln 2/t_(1/2) and t_(1/2) is the half life for²²⁴Ra.

The radiation dose, D, is defined as energy per mass, i.e., J/kg inSI-units.

For the complete decay of 1 MBq of ²²⁴Ra in 1 ml of aqueous liquid itwould amount to D=(4.53×10¹¹×28 MeV×1.6×10⁻¹³ J/MeV)/10⁻³ kg=2029 Gythat is, in one half life of 3.64 days a solution of 1 MBq/ml of ²²⁴Rain equilibrium with daughters will be exposes to about 1 kGy ofself-irradiation. For a monoclonal antibody it is usually advisable bekeep the self dose of the radiopharmaceutical solution below 0.5 kGy toavoid reduced binding properties due to radiolysis.

Thus, a kit system whereby chelator conjugated antibody is added, e.g.,by a syringe to a vial containing the ²²⁴Ra (including daughters)solution a few hours to a few minutes before the product is administeredto a patient is advised for concentrated solutions of ²²⁴Ra withprogenies suitable for long distance shipment.

Example of scavenging ²¹²Pb with TCMC-monoclonal antibody conjugate in a7 days old ²²⁴Ra solution. A ²²⁴Ra solution produced one week earlierwas added 10% ammonium acetate and TCMC-rituximab (to a finalconcentration of about 5 mg/ml) to a final volume and pH of about 0.1 mland 5.5, respectively, and kept at room temperature overnight. After 18hours a sample was withdrawn and mixed with formulation buffer asdescribed. TLC analyses showed that 91% of the ²¹²Pb activity wasprotein bound, i.e., in the lower half of the TLC strip as determined bygamma counting. This demonstrates that a kit with a vial containing a²²⁴Ra solution and a separate vial with a chelator-labeled protein orsimilar can be combined and used to scavenge ²¹²Pb in the ²²⁴Ra severaldays after ²²⁴Ra production date, thereby preparing a ²²⁴Ra bone-seekerwith a ²¹²Pb tumor-seeking complex using short reaction times to avoidradiolytic degradation of the product.

It was verified that the reagents, other than ²²⁴Ra, could be storedseveral weeks or months without losing their function, thus, they arewell suited for use in a kit format.

Example 11 Circulating Tumor Cells

Circulating tumor cells could give rise to new tumor lesions in bone orsoft tissues and may be addressed by the novel radiopharmaceuticalsolution described herein.

One may argue that the amount of ²¹²Pb in therapeutic ²²⁴Ra solution maybe moderate to modest (i.e., at equilibrium about 1.1 times that of²²⁴Ra). If one assume similar dosing of ²²⁴Ra as is done with ²²³Ra inpatients but correct for the half life difference, roughly 150 kBq perkg of bodyweight would be the administered dose. This is just an exampleand a dosing may differ significantly according to disease and whatlevel of side effects that is acceptable.

At equilibrium 150 kBq per kg of body weight would translate into a²¹²Pb-antibody conjugate dosage of about 11.5 MBq in 5 litre of blood ina 70 kg patient. The number of circulating tumor cells is typically lessthat 10 cells per ml thus in 5 l blood there are less than 50 000 tumorcells in total. If only 1 in 100 000 of the injected ²¹²Pb-antibodyconjugate binds to the tumors cells this would mean 0.0023 Bq per cell,equivalent to 127 ²¹²Pb atoms bound per cell, which would be highlydestructive as it has been reported that a mean of 25 cell bound ²¹²Pbper cell would kill 90% of a cell population.

The number of atoms bound to a cell will depend on the specific activityof the ²¹²Pb-conjugated antibody and the number of antigens available atthe target cells. Lead-212 labeled TCMC-trastuzumab with a specificactivity of about 37 MBq/mg (1 mCi/mg) was recently evaluated in aclinical study.

A ²¹²Pb-labeled monoclonal antibody with a specific activity of 37 MBqper mg has a ²¹²Pb atom to antibody molecule radio of 1:1973, that is,very few of the antibody molecules are actually radiolabeled. To reach a90% cell kill level of ²¹²Pb of 25 atoms per cell, one would need tobind 49325 antibody molecules per cell. This is obtainable as severaltumor associated antigens are expressed at levels exceeding this. Alsofrom a chemical stand point this is plausible as it is possible toconjugate typically 1 to 5 chelator units per antibody molecule withoutlosing the antigen binding properties, so a very small fraction of thechelator-groups are actually occupied by ²¹²Pb during and after theradiolabeling.

REFERENCES

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1. A radiopharmaceutical solution comprising a free uncomplexed ²²⁴Ra insolution with ²¹²Pb complexed with EDTMP, antibody-conjugated-DOTA, orantibody-conjugated-TCMC.
 2. (canceled)
 3. The radiopharmaceuticalsolution according to claim 1, wherein ²¹²Pb is complexed with aTCMC-conjugated monoclonal antibody. 4.-5. (canceled)
 6. A method ofinhibiting a skeletal disease comprising: administering theradiopharmaceutical solution according to claim 1 to a subject that hasa skeletal disease, wherein the skeletal disease presents skeletalmetastases from cancers to the breast, prostate, kidneys, lung, bone,thyroid, multiple myeloma or primary skeletal cancer, or osteosarcoma,whereby the radiopharmaceutical solution inhibits said skeletal disease.7. (canceled)
 8. A method of making a radiopharmaceutical solution ofclaim 1 comprising: mixing a solution comprising free uncomplexed ²²⁴Rawith EDTMP, antibody-conjugated-DOTA, or antibody-conjugated-TCMC. 9.The radiopharmaceutical solution according to claim 1, wherein ²¹²Pb iscomplexed with EDTMP.
 10. The radiopharmaceutical solution according toclaim 1, wherein ²¹²Pb is complexed with a DOTA-conjugated monoclonalantibody. 11.-12. (canceled)
 13. The radiopharmaceutical solutionaccording to claim 1, wherein the complexing agent is (Herceptin)TCMC-trastuzumab.
 14. The radiopharmaceutical solution according toclaim 1, wherein the solution is in a volume of 100 μL to 10 mL and theradioactivity is 100 kBq to 100 MBq.
 15. (canceled)